Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from its highly ordered structure, the detailed mechanism by which it functions has proven elusive. This is because proteins are not simply static macromolecules that host an active site, as depicted by their crystal structure;rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this choreography of life at the atomic level with ps time resolution. This choreography of life is being investigated by time-resolved techniques that are based on the pump-probe method. The pump is usually a laser pulse that triggers, at a well-defined instant of time, the process we wish to investigate. The duration of the laser pulse can be as short as a few tens of femtoseconds, which allows us to access the chemical time scale for molecular motion. After photoexcitation, a time-delayed probe pulse is directed through the pump-illuminated volume to interrogate the system. When the probe pulse is derived from an X-ray source, we can probe the protein structure via Laue diffraction or via Small- and Wide-Angle-X-ray-Scattering (SAXS/WAXS). When the probe pulse is generated in the uv-vis or mid-IR region, we can interrogate the system spectroscopically. The time resolution of the pump-probe method is limited only by the duration of the pump and probe pulses and the timing jitter between them. Each pump-probe measurement produces a time-resolved snapshot of the protein. By stitching together a series of snapshots, we create a movie that, in the case of time-resolved Laue diffraction, provides a near-atomic view of the correlated structure changes triggered by the pump pulse. We can literally watch a protein as it functions! Time-resolved Laue studies are performed using protein crystals. The intermolecular forces that maintain crystalline order give rise to high resolution diffraction spots, but those same forces constrain large amplitude conformational motion. This loss of conformational flexibility may perturb, and may even inhibit the function of a protein. Nonetheless, this method stands alone in its ability to acquire near-atomic structural information on ultrafast time scales. X-rays can also extract structural information from molecules in solution where the full range of conformational motion is permitted. Because there is no long range order in protein solutions, X-ray scattering from the protein is diffuse, but contains diffraction rings that provide a fingerprint that can be correlated with the protein structure. Though the information provided by time-resolved SAXS/WAXS is not at atomic resolution, models of the protein structure can be compared against the measured scattering spectrum, and time-dependent changes of the SAXS/WAXS fingerprint can be used to assess putative models that describe the reaction pathway. Our efforts to develop time-resolved X-ray methods to study protein function are summarized in separate annual reports. Since these studies all use light as a trigger, pursuing these studies requires detailed knowledge of the photophysics of proteins. Moreover, we need to be able to assess the dynamics for the time-ordered sequence of events that follow the laser trigger. Finally, we need to be able to make these measurements in protein solutions as well as crystals. Time-resolved spectroscopy is well suited for such studies, and is the focus of this annual report. We have developed an ultrafast time-resolved microfocusing spectrophotometer that is capable of probing protein dynamics in solution and in crystals. The probe pulses used in this home-built spectrometer are generated by an Optical Parametric Amplifier (OPA) whose signal output is used to produce a broadband, single filament, white-light continuum through nonlinear interactions with a transparent dielectric medium. The white-light continuum has a broad spectral range that spans from 375 to 1000 nm. The pump pulse used to photoexcite the sample can come from multiple laser sources. A second OPA is used to generate femtosecond tunable pulses that can be delayed out to a few ns. An Optical Parametric Oscillator (OPO) with broad tunability throughout the visible generates 2-3 ns pulses that can be delayed out to seconds. A Q-switched frequency-doubled Nd:YAG laser is used to generate 200 ns pulses at 532 nm that can be delayed out to seconds. A CW 527 nm laser can be gated on and off with an AOM that provides approximately 200 ns rise/fall times. An electronic timing-system was developed to properly synchronize the various pump sources to the probe pulses. We have used this time-resolved microfocusing spectrometer to study ligand dynamics in myoglobin (Mb) and hemoglobin (Hb) as well as mutants of both. One of the challenges in studying tetrameric Hb is the number of possible species generated when the photolysis is incomplete. To generate a more homogenous photolyzed state, we pumped the sample with an intense 200 ns, 532 nm pulse, which is long compared to the time scale for Hb rotation and geminate recombination, thereby ensuring efficient and complete photolysis. This source has been combined with the AOM-gated source to maintain the photolyzed state. The broad continuum source provides access to the Soret band, the Q-band, and the near-IR bands, including band III. Because the Soret band absorbance is strong, studies in that region of the spectrum require dilute samples. When investigating samples at concentrations suitable for time-resolved X-ray studies, the protein concentration is too high to track the Soret band. Consequently, those studies have focused on the Q-band and band III. Our spectrometer records a complete absorption spectrum with a single 100-fs duration probe pulse. Time-resolved spectra are generally acquired on a logarithmic sequence of pump-probe time delays, and their evolution informs us about the ligation state of the heme as well as the tertiary and quaternary state of the protein. To extract the maximum amount of information contained within our time-resolved spectra, we continue to develop novel approaches for analyzing time-resolved data. We recover via nonlinear least-squares methods the rate coefficients that describe the population dynamics of all states invoked by our model;at the same time, we extract via linear least-squares methods the time-independent basis spectra for those states. Because we solve the population dynamics problem by integrating differential equations on a logarithmic timescale, we are able to account for the saturating effects of high fluence pump pulses used in time-resolved X-ray studies. Through a combination of time-resolved optical and X-ray studies, were now able to investigate allostery in proteins at an unprecedented level of detail. The analysis of data acquired thus far is ongoing, but preliminary analysis suggests that we should be able to extract from our data the relative R-T reaction rates for different ligation states. Due to laser failures (femtosecond oscillator and pump laser for the regenerative amplifier), we have not yet had an opportunity to follow up with those studies.